Lysophosphatidic acid (LPA) is a kind of glycero-phospholipids with a lipid bonded to one of the hydroxyl groups in the glycerin structure. LPA is a substance generated from glycerophospholipid, where one of two lipid moieties has been hydrolyzed through the intervention of phospholipase. As representative examples of LPA, such as 1-acyl-glycerol-3-phosphate, 2-acyl-glycerol-3-phosphate and 1-alkenyl-glycerol-3-phosphate are known.
LPA, present only in trace amounts in vivo, has been recognized merely as intermediate or resolvent occurring during biosynthesis of glycerophospholipid. Recently however, it has been revealed that LPA is a primary component of lipid growth factor present in serum (see Prior Art Reference 22), having a variety of physiological activities, and thus has attracted the public attention as one of physiologically active lipids.
LPA is found to be a substance that stimulates cell proliferation, and is indispensable for recovery of the damaged tissue. It has also been reported that LPA induces retraction of neurite which is essential to the maturation of neuron, and carcinoma cell invasion. Moreover, LPA has come to be known to have a variety of functions in diverse tissues such as smooth muscle contraction, platelet aggregation, suppression of cell apoptosis and promotion of cellular chemotaxis. Further, a number of patent applications relating to “The method of detecting the ovarian cancer by detecting LPA in plasma” (see Prior Art Reference 1), “The method for determining the level of lysophosphate acid such as LPA in a sample” (see Prior Art Reference 2), “The method of producing Lysophosphatidate Phosphatase which specifically hydrolyzes LPA” (see Prior Art Reference 3) and “Cloning of human Lysophosphatidate Phosphatase” (see Prior Art Reference 4) have been filed.
As described above, it has been revealed that LPA is not merely an intermediate or resolvent but itself is a kind of mediator having a variety of physiological activities. Accordingly, LPA, same as a platelet activating factor (PAF) and sphingosine-1-phosphate (S1P), has come to be recognized as one of “lysophospholipid mediators”.
It has been recognized that LPA exhibits its physiological activities by binding to G-protein-coupled receptor (GPCR) expressed on the surface of the cell membrane. Namely, the receptor of LPA has been recognized as a seven transmembrane GPCR, however, remained substantially unknown over a long period of time. It is because, LPA being a kind of lysophospholipids and fat-soluble, even the existence of its receptor has been doubted due to the difficulties in carrying out membrane binding assay or in finding a suitable antagonist.
However, in 1996, genes of LPA receptor named vzg-1 (see Prior Art Reference 23) and PSP24 (see Prior Art Reference 24) were cloned. It was named vzg-1 (ventricular zone gene-1). It is because vzg-1, a seven transmembrane GPCR specific to the cell in the neocortical ventricular zone, where interkinetic nuclear movement of neuroblast harmonious to cell progression cycle has been observed during the developmental process of the mouse brain. To date, vzg-1 was identified as a mouse homologue of edg-2 gene, which has been isolated as a sheep-derived orphan receptor, and is now known as edg-2 gene.
In addition to edg-2 (vzg-1), a number of genes exhibiting a high degree of sequence identity such as edg-1, edg-3 and edg-4 have been recorded in EST database, and recognized to form a group of EDG (endothelial cell differentiation gene) family. EDG family divides into two subgroups according to their homology, one is a group of edg-2 and edg-4 which functions as LPA receptor, another is a group of edg-1 and edg-3 which functions as S1P (sphingosine-1-phosphate) receptor.
PSP24 gene exhibits fairly little sequence homology to the above described edg-2 gene, however, the expression of the homologous gene to PSP24 has been observed in mouse nerve system and human fatal brain. Since the gene products thereof also exhibit reactivity with LPA, it is considered to form an independent group. Human PSP24 gene also has been isolated from adult human brain cDNA library (see Prior Art Reference 5) based on the base sequence of PSP24 gene of Xenopus laevis oocyte.
EDG family and PSP24 being members of GPCR, the known GPCRs to date are shown in FIG. 1. FIG. 1 is a phylogenetic tree, wherein the location of each protein has been determined based on the homology in amino acid sequence thereof, and those exhibiting a higher degree of sequence homology are placed in the vicinity while those exhibiting lower degree of sequence homology have been placed in a way off. The numbers in FIG. 1 represent measurement for the relative value indicating lower degree of sequence homology between individual proteins. Closed circles (●) in FIG. 1 represent the known receptors for lipids, whereas gray circles represent the receptors known for other than lipids. Generic names of ligands are displayed in outskirt of the tree. EDG family is located in the lower right in FIG. 1. And PSP24 is located close to the lower center. Open circles in FIG. 1 represent the orphan receptors whose ligands have not been identified yet.
p2y9, a GPCR of the present invention, is located in the lower left in FIG. 1. Thus, it is apparent from FIG. 1 that p2y9 shares no homology in amino acids sequences with known EDG family or PSP24.
The novel GPCRs are being identified even now, and disclosed in the published patent applications such as Japanese Unexamined Patent Publication No. 2002-355045 (see Prior Art Reference 6), Japanese Unexamined Patent Publication No. 2002-17378 (see Prior Art Reference 7) and other patent applications (see Prior Art References 8 to 18). The patent applications relating to the method for modifying the function of GPCR (see Prior Art Reference 19), the method for regulating transcription of G2A receptor (see Prior Art Reference 20), and the method for screening the activity of GPCR (see Prior Art Reference 21) have been filed.
Prior art references relating to the present invention are shown below, and those are incorporated herein by reference.    1. Japanese Unexamined Patent Publication No. 2002-328132    2. Japanese Unexamined Patent Publication No. 2002-017398    3. Japanese Unexamined Patent Publication No. 2000-152782    4. Domestic re-publication of PCT international publication WO00/031275    5. Domestic re-publication of PCT international publication WO99/024569    6. Japanese Unexamined Patent Publication No. 2002-355045    7. Japanese Unexamined Patent Publication No. 2002-17378    8. Japanese Unexamined Patent Publication No. 2001-245674    9. Japanese Unexamined Patent Publication No. 2001-245673    10. Japanese Unexamined Patent Publication No. 2001-245672    11. Japanese Unexamined Patent Publication No. 2001-211889    12. Japanese Unexamined Patent Publication No. 2001-190281    13. Japanese Unexamined Patent Publication No. 2001-186888    14. Japanese Unexamined Patent Publication No. 2001-169786    15. Japanese Unexamined Patent Publication No. 2001-161385    16. Japanese Unexamined Patent Publication No. 2001-161383    17. Japanese Unexamined Patent Publication No. 2001-161382    18. Published Japanese translation of a PCT application No. 2002-501083    19. Published Japanese translation of a PCT application No. 2002-523091    20. Published Japanese translation of a PCT application No. 2001-523456    21. Published Japanese translation of a PCT application No. H11-505718/1999    22. van Corven E., et al., Cell, 59, 45-54 (1989)    23. Hecht, J. H., et al., J. Cell. Biol., 135, 1071-1083 (1996)    24. Guo, Z., et al., Proc. Natl. Acad. Sci. USA, 93, 14367-14372 (1996)